Studies on the Characterization and Distribution of Fatty Acids and Minor Components of High-Erucic Acid Mustard Oil and Low-Erucic Acid Rapeseed Oil

Emir. J. Food Agric. 2012. 24 (4): 281-287 http://ejfa.info/

NUTRITION AND FOOD SCIENCE Studies on the characterization and distribution of fatty acids and minor components of high-erucic acid mustard oil and low-erucic acid rapeseed oil

Md. Abdul Alim1,2, Zafar Iqbal2* and Paresh C. Dutta1

1Department of Food Science, Uppsala BioCenter, Division of Food Chemistry, Swedish University of Agricultural Sciences (SLU), P. O. Box 7051, SE-750 07, Uppsala, Sweden 2Department of Food Technology and Rural Industries, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Abstract

The aim of this work was to compare the positional fatty acid composition, sterols, tocopherols and oxidative stability of mustard oil (MO) and rapeseed oil (RSO). The MO contained higher levels of erucic acid (42.8%) and linolenic acid (18.2%), compared with RSO (0.4 and 8.6%) whereas the RSO contained higher levels of oleic acid (62.2%) and linoleic acid (19.5%), than that of MO (17.4 and 15.8%). The changing fatty acid compositions at different position led to different physical properties. The ω-6/ω-3 ratio of MO and RSO were 0.87 and 2.27, respectively. The balance of ω-6/ω-3 ratio is also an important determinant in decreasing the risk for coronary heart disease. MO contained higher amount of total tocopherols (38.32 mg/100g) but lower amount of total sterols (606.32 mg/100g) than that of RSO (631.98 mg/100g and 25.57 mg/100g). The oxidative stability determined by Rancimat test of MO (PF, 1.57) was higher compared with RSO (PF, 1.0).

Key words: Mustard oil, Rapeseed oil, Fatty acids, Sterols, Tocopherols, Oxidative stability

Introduction Lipids (oils and fats), apart from providing (allyl isothiocyanate), hot and nutty taste. nutrition, are an integral part of human diets Bangladeshi consumers like such strong smell, hot throughout the world, which are products from nutty taste, pungent and sulfurous odor. MO contains plants and animals kingdom with great variation in fatty acids like erucic, oleic, linoleic and α-linolenic the composition of fatty acids that are of acid. This oil contains a little less than 60% of importance for both the chemical and the food monounsaturated fatty acids out of which 42% industries. Lipids are known to play functional erucic acid and 12% oleic acid. It contains 21% roles in contributing to the palatability of processed polyunsaturates (6% ω-3 α-linolenic acid and 15% foods which provide human nutrition. Actually, ω-6 linolenic acid) and it has 12% saturated fats consumers tend to prefer oil which is available in (USDA National Nutrient Database). It contains the their region. In Bangladesh, commonly used edible pungent Allyl isothiocyanate and has about 60% oils are soybean and mustard oil (MO). In India and monounsaturated fatty acids of which 42% erucic Bangladesh, MO is collected from Mustard seeds acid and 12% oleic acid, it has 21% polyunsaturates by traditional way. of which 6% is the omega-3 alpha-linolenic acid and MO makes up about 24-40% of the mustard 15% omega-6 linoleic acid and it has 12% saturated seeds, which is characterized by the presence of fats (USDA National Nutrient Database). MO may higher level of erucic acid and it has the lowest provide a protective effect in patients with acute saturated fatty acids content among all the edible myocardial infarction possibly due to the presence of vegetable oils. MO has a strong smell, pungent odor α-linolenic (Singh et al., 1997). However, higher levels of erucic acid are unsuitable for human consumption as food purposes (Kramer et al., 1982). Received 30 January 2012; Revised 19 February 2012; Generally, erucic acid enriched oil is useful for the Accepted 21 February 2011 polymer industry and is valuable raw material for *Corresponding Author manufacture of industrial products such as Zafar Iqbal plasticizers, detergents, surfactants, polyesters and Department of Food Technology and Rural Industries, coatings whereas oils low in erucic acid are Bangladesh Agricultural University, Mymensingh-2202, recommended for food purposes because oils high in Bangladesh erucic acid may cause an accumulation of Email: [email protected]

281 Md. Abdul Alim et al. triacylglycerol in the hearts of animals (Beare- retention of sterols and tocopherols, and the Rogers et al., 1971). Higher consumption of erucic oxidative stability of MO and RSO. acid may increase the concentration of adrenal Materials and Methods cholesterol causing fibrotic changes in myocardium, Oils and chemicals liver weight and cholesterol (Aaes-Jorgensen, 1972; Mustard oil (MO) was supplied from Beare-Rogers et al., 1972). High levels of erucic acid Agricultural Marketing Co. Ltd. (Dhaka, is not suitable for human food in European Union Bangladesh). Rapeseed oil (RSO) was from AAK and other developed countries since erucic acid (Aarhus Karlshamn AB, Malmö , Sweden). The showed serious pathological changes in the heart and heptadecanoic acid, acetic acid and pancreatic skeletal in animals (Food Standards Australia New lipase from porcine pancreas (Type II) were Zealand, 2003). obtained from Sigma-Aldrich Chemical Co. (St. Rapeseed is the third leading source of edible oil Louis, MO, USA). Standard of sterols 5α- in the world. Rapeseed oil (RSO) is the most useful cholestane was obtained from Steraloids (Newport, of all cooking oils and it contains a significant RI, USA), reference samples of tocopherols were amount of n-3 and n-6 fatty acids. RSO contains obtained from Merck (Darmstadt, Germany) and mostly of the fatty acid such as oleic, linoleic, Tri-Sil reagent was from Pierce Chemical Co. linolenic, palmitic and strearic acid (Gunstone, et al., (Rockford, IL, USA). The standard mixture of fatty 1994; Hui, 1996). The oil content usually makes up acid methyl esters (FAME) F-07 was from Larodan about 40-60% of rapeseed. RSO consists 95% of Fine Chemicals AB (Malmö , Sweden). All other triacylglycerols (TAG) and 5% non-triacylglycerols, chemicals and solvents were from VWR known as minor components like free fatty acids, (Stockholm, Sweden), unless otherwise stated. mono- and diacylglycerols, phospholipids, tocopherols, tocotrienols, flavonoids, other phenolic Hydrolysis by pancreatic lipase for positional compounds, pigments (chlorophylls), sterols etc fatty acid composition (Shahidi and Shukla, 1996). The MO and RSO (8 mg) were taken in a test Every vegetable oil has its own stability against tube. Eight mL of Tris-HCl buffer (pH 8.0), 2 mL oxidation depending on the fatty acid compositions of 0.05% bile salt in distilled deionized water (w/v), and the content of antioxidants which it contains 0.8 mL of 2.2% CaCl2 in distilled deionized water (Nogala-Kalucka et al., 2005; Kamal-Eldin, 2006; (w/v) and 20 mg of pancreatic lipase (porcine Przybylski and Eskin, 2006). Figure 1 shows the pancreatic lipase, crude type II) were mixed for structure of linoleic (polyunsaturated fatty acid) and hydrolysis and vortexed for 30 sec. The mixture erucic acid. was incubated in a water bath at 40°C for 3 min, 1 mL of diethyl ether was added. Diethyl ether was evaporated under nitrogen gas. The hydrolytic (a) products were separated on thin-layer chromatography (TLC) plate (silica gel 60, 20x20 (b) cm, 0.25 mm thickness, Merck, Eurolab AB, Stockholm, Sweden) by developing solvent of Figure 1. (a) Chemical structure of polyunsaturated fatty hexane/diethyl ether/acetic acid (50/50/1, v/v/v). acid (linoleic acid); (b) erucic acid (Wikipedia, 2012). The band of monoacylglycerol was scrapped off for methylation and analyzed by GC. After that, the Generally, polyunsaturated fatty acids are percentage of fatty acid at sn-1,3 position was responsible for oxidation and off-flavours. calculated by the following formula : Sn-1,3 (%) = However, antioxidant compounds present in oil (3 T – sn-2)/2 where T is the total fatty acid which is important in the prevention and treatment contents of MO and RSO, respectively. All of diseases such as heart disease, autism, cancer, analyses were conducted in triplicate and GC stroke, diabetes, Alzheimer’s dementia, Parkinson’s analysis was done as described above. disease, arthritis and muscular degeneration (Manna et al., 2002). It also contributes to lowering Fatty acid composition serum cholesterol levels in human body (Moreau, The triacylglycerol (TAG) fraction in the MO 2004) also to the oxidative stability and shelf-life of and RSO were separated by TLC plate (silica gel oil (Przybylski and Eskin, 2006). 60, 20x20 cm, 0.25 mm thickness, Merck, Eurolab The main objectives of this research work were AB, Stockholm, Sweden) developed with to study the positional fatty acids composition, hexane/diethyl ether/acetic acid (85/15/1, v/v/v). The visualized band corresponding to TAG

282 Emir. J. Food Agric. 2012. 24 (4): 281-287 http://ejfa.info/ molecule was scraped off into a screw-capped tube B.02.01 (Agilent Technologies, Wilmington, DE, and dissolved in 0.5 mL hexane. 3 mL 0.01 M USA) connected with a GC PAL auto sampler NaOH in dry methanol was added into the tube. (CTC Analytics AG, Zwingen, Switzerland) and a Fifty μL heptadecanoic acid (C17:0, 1 mg/mL in flame ionization detector. A combination of two hexane) as an internal standard was also added in fused-silica capillary columns, a DB-5MS (length the test tube. The test tube was closed with a 15 m, i.d. 0.18 mm and film thickness 0.18 μm) and stopper and vortexed for proper mixing. The a DB-35MS (length 10 m, i.di 0.2 mm, and film sample was incubated in water bath at 50°C for 30 thickness 0.33 μm) were used which were joined min under continuous shaking. Two mililiters (50% together by a universal press-fit connector. The NaHSO4: 25% NaCl in water) solution were added temperatures of detector and injector were 310 and and cool under running water. Three milliliters 260ºC, respectively. The carrier gas was helium and water and 1 mL hexane were added and vortexed nitrogen was used as make-up gas at flow rates of vigorously. The hexane layer was separated and 0.7 and 30 mL/min, respectively. The initial oven solvent was evaporated with nitrogen. Gas temperature was 60ºC for 1 min and increased to chromatography (GC, Chrompack CP 9001, 290ºC at a rate of 50 ºC per min and maintained for Middelburg, The Netherlands), accompanied with 5 min and then increased again by 1ºC per min to auto-injection and flame-ionization detection was 305ºC for 15 min. All analyses were conducted in used for fatty acid composition analysis. A 50 m X triplicates and quantification was performed using 0.22 mm, 0.25 μm film thickness fused-silica 5α-cholestane an internal standard. capillary column BPX70 (SGE, Austin, TX, USA) Analysis of tocopherols by High-Pressure Liquid was used for separation. Injector and detector Chromatography (HPLC) temperatures were 240 and 280C, respectively. Quantitative tocopherols content were analyzed Oven conditions were 160C increased to 220C at by HPLC. The HPLC system consisted of a 510 a rate of 2C/min and maintained for 5 min. The HPLC pump (Watres, Milford, USA) and carrier gas was helium and nitrogen as a make-up Rheodyne Injector with 20 μL loop as well as a gas at a flow rate of 30 mL/min. Fatty acid methyl fluorescence detector Varian 9070 (Walnut Creek, esters (FAME) were identified by comparison of CA, USA). The detector was set at the wavelength their retention time with standared FAME. The of 294 and 320 nm for excitation and emission, peak areas were integrated by maestro version 2.4 respectively. The column was LiChroCART 250-4 (Chrompack Middelburg, The Netherlands) and (Merck KGaA, Darmstadt, Germany) packed with reported as percentage of the total fatty acids Lichrosphere 100 NH2, 5 μm particle size, and (Azardmard-Damirchi and Dutta, 2008). All coupled to a LiChroCART 4-4 guard column analyses were conducted in triplicate. (Merck KGaA, Darmstadt, Germany). Analysis of sterols by GC Approximately, 10 mg of each sample were The MO and RSO (20 mg) were taken in a test dissolved in 1 mL n-heptane and 20 μL were tube and added 1 mL 2M KOH in ethanol (95%). injected manually. The isocratic mobile phase was The tubes was placed in a boiling water bath for 10 a mixture of n-heptane: tert-butyl methyl ether. min with intermittent shaking and thereafter cooled tetrahydrofuran: methanol (79:20:0.98:0.02, under running water. Thereafter, 1 mL water, 2 mL v/v/v/v) at the flow rate of 1.0 mL/min. Each hexane containing 20µg 5α-cholestane as internal tocopherol was quantified using an external standard and 200 µL absolute ethanol were added, standard method and the area of each peak was shaked vigorously and centrifuged. The hexane calculated by an HP 3396A integrator (Hewlett- layer was transferred to a small test tube and Packard, Avandale, PA, USA) (Azardmard- evaporated the solvent fully under a stream of Damirchi and Dutta, 2008). All analyses were nitrogen. For derivatization, 100 μL Tri-Sil conducted in triplicate. reagents was added and dispersed by brief Rancimat test for oxidative stability sonication. Then, the sample was incubated at 60ºC The induction period, measuring the increase in for 45 min and dispersed the reagent in the the volatile by-products released from the oxidizing ultrasonic bath. The solvent was evaporated under a oil of MO and RSO was determined by the stream of nitrogen. The TMS- ether derivates of the rancimat method (Rancimat 743, Metrohm, sterols were dissolved in 500 μL hexane. About 1 Switzerland) at 100ºC with the air flow rate of 25 μL sample was injected in splitless mode on GC L/h. The conductivity was measured for estimating model 6890 and software ChemStation Rev. the concentration of the degradation products and

283 Md. Abdul Alim et al. longer induction period showed higher oxidative levels of palmitic acid (16:0) ranged from 4.1 to stability. The protection factor (PF), which is 4.9% were found in MO and RSO. calculated by dividing the induction period either of The RSO contained higher levels of oleic acid the MO or RSO by that of RSO (induction time, 2.2 (18:1, 54.0%) at sn-2 position than that of MO h) was obtained as the relative activity (Schwarz (18:1, 35.3%), whereas at sn-2 position, the MO and Ernst, 1996). contained higher levels of linoleic acid (18:2, 39.4%) and linolenic acid (18:3, 22.7%) than that of Results and Discussion RSO (18:2, 32.4% and 18:3, 13.5%, respectively). Total and positional fatty acid compositions The presence of higher amount of unsaturated fatty The total and positional fatty acid compositions acids at sn-2 position is important nutritionally, of MO and RSO are presented in Table 1. because it is easily converted during digestion and The MO and RSO contained total unsaturated absorbed in the body (Quinlan and Moore, 1993). fatty acids 94.2 and 92.3% as well as total saturated The fatty acid contents at sn-1,3 position were fatty acids 5.8 and 7.7%, respectively. The MO erucic (22:1, 63.5%), linolenic (18:3, 15.9%), oleic contained a slightly higher amount of total (18:1, 8.5%), palmitic (16:0, 5.8%) and linoleic unsaturated fatty acid (94.2%) than RSO (92.3%). (18:2, 4.0%), respectively for MO. In case of RSO, The major fatty acids of MO were erucic (22:1), the fatty acid contents at sn-1,3 position were oleic linolenic (18:3, ω-3), oleic (18:1) and linoleic acid (18:1, 66.3%), linoleic (18:2, 13.1%), palmitic (18:2, ω-6) which composed of 42.8, 18.2, 17.4 and (16:0, 7.3%) and linolenic (18:3, 6.2%), 15.8%, respectively. On the other hand, the RSO respectively. Such changes of fatty acid contained oleic (18:1), linoleic (18:2, ω-6) and compositions at different position lead to different linolenic acid (18:3, ω-3) which composed of 62.2, physical properties. The ω-6/ω-3 ratio (n-6/n-3 19.5 and 8.6%, respectively. The MO contained ratio) of MO and RSO were 0.87 and 2.27, higher levels of erucic acid (42.8%) and linolenic respectively. Healthy ratios of ω-6:ω-3 range from acid (18.2%), respectively as compared with RSO 1:1 to 1:4. An individual needs more ω-3 than ω-6 (0.4 and 8.6%, respectively) whereas the RSO (Tribole, 2007; Lands, 2005). The balance of ω- contained higher levels of oleic acid (62.2%) and 6/ω-3 ratio is also an important determinant in linoleic acid (19.5%), respectively than that of MO decreasing the risk for coronary heart disease both (17.4 and 15.8%, respectively). Almost similar in the primary and secondary prevention of coronary heart disease (Simopoulos, 2002).

Table 1. Positional fatty acids composition (% ±SD) of mustard oil (MO) and rapeseed oil (RSO). All analytical results are presented as mean and standard deviation of triplicate measurement (n = 3). Fatty acids MO RSO sn-1,3 sn-2 Total sn-1,3 sn-2 Total 16:0 5.8±0.1 0.8±0.05 4.1±0.1 7.3±0.12 0.1±0.05 4.9±0.1 16:1 nda nd nd 0.3±0.0 nd 0.2±0.0 18:0 2.3±0.3 0.4±0.05 1.7±0.2 2.7±0.07 nd 1.8±0.05 18:1 8.5±0.2 35.3±0.3 17.4±0.2 66.3±0.1 54.0±0.5 62.2±0.1 18:2 (ω-6) 4.0±0.1 39.4±0.1 15.8±0.1 13.1±0.05 32.4±0.2 19.5±0.1 18:3 (ω-3) 15.9±0.2 22.7±0.2 18.2±0.15 6.2±0.02 13.5±0.2 8.6±0.05 20:0 nd nd nd 0.9±0.07 nd 0.6±0.05 20:1 nd nd nd 2.1±0.15 nd 1.4±0.1 22:0 nd nd nd 0.5±0.2 nd 0.3±0.05 22:1 63.5±0.4 1.4±0.1 42.8±0.3 0.6±0.15 nd 0.4±0.1 24:0 nd nd nd 0.2±0.2 nd 0.1±0.05 ∑SFAb 8.1 1.2 5.8 11.6 0.1 7.7 ∑UFAc 91.9 98.8 94.2 88.4 99.9 92.3 ω-6/ω-3 ratio 0.87 2.27 Oxidative stabilityd 1.57 1.0 a nd, not detected b Total sum of saturated fatty acid c Total sum of unsaturated fatty acid d Values expressed as protection factor (PF). PF was calculated by dividing the induction period either of the MO or RSO by that of RSO (induction time, 2.2 h)

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Table 2. Sterols and tocopherols content (mg/100g±SD) of mustard oil (MO) and rapeseed oil (RSO). All analytical results are presented as mean and standard deviation of triplicate measurement (n = 3). Sterols and Tocopherols MO RSO Cholesterol 2.03±0.6 7.01±0.31 Brassicasterol 72.11±0.9 59.24±1.66 Campesterol 203.60±2.31 238.86±3.39 Stigmasterol 3.27±0.25 nd Sitosterol 307.71±4.12 323.90±5.90 Δ5-avenasterol 17.60±.22 2.97±0.31 Total 606.32 631.98 α-tocopherol 5.15±0.28 3.40±0.3 β-tocopherol 0.71±0.15 nd γ-tocopherol 30.46±0.54 19.44±0.78 δ-tocopherol 2.0±0.29 2.73±0.2 Total 38.32 25.57

Sterols and tocopherols content of mustard oil fats, as it gives a good estimation of their (MO) and rapeseed oil (RSO) susceptibility to oxidative degradation (Aparicio et The distribution of sterols and tocopherols for al., 1999). The results of rancimat test are presented MO and RSO were assessed. The contents of in Table 2. A higher protection factor (1.57) for sterols and tocopherols (mg/100g) of MO and RSO MO was observed compared to that of RSO (1.0). are presented in Table 2. Generally, higher protection factor suggests The contents of cholesterol, brassicasterol stronger oxidative stability. The difference in campesterol, stigmasterol, sitosterol and Δ5- protection factor of MO was mainly due to different avenasterol were 2.03, 72.11, 203.6, 3.27, 307.71 levels of total tocopherols content. and 17.6 mg/100g, respectively) in MO. The RSO Conclusions contained higher amount of sterols (631.98 Mustard oil is very popular to the Bangladeshi mg/100g) than that of MO (606.32 mg/100g). The people due to its strong smell, hot nutty taste, RSO contained cholesterol (7.01 mg/100g), pungent and sulfurous odor. This oil contains brassicasterol (59.24 mg/100g), campesterol higher levels of erucic acid which is not suitable for (238.86 mg/100g), sitosterol (323.90 mg/100g) and human consumption as food purposes but it has Δ5-avenasterol (2.97 mg/100g), respectively. The higher oxidative stability (due to its high content of major sterol in RSO was sitosterol followed by antioxidants) compared to RSO. Oxidative stability campesterol, brassicasterol, cholesterol, and Δ5- is an important criterion in ascertaining the quality avenasterol. The contents of cholesterol, of oils and fats. The balance of ω-6/ω-3 ratio (1:1 to campesterol and sitosterol (7.01, 238.86 and 323.9 1:4) is also an important determinant in decreasing mg/100g, respectively) in RSO were higher than the risk for coronary heart disease which was that of MO (2.03, 203.6 and 307.71 mg/100g, determined in a good range in the case of MO respectively). On the other hand, the content of (0.87). Future works are to be designed in our brassicasterol and Δ5-avenasterol (72.11 and 17.6 laboratory for the extraction of erucic acid from mg/100g, respectively) were higher in MO than that MO to make this valuable and nutritious vegetable of RSO (59.24 and 2.97 mg/100g, respectively). oil more useful as a food product for human Vegetable oils are good sources of tocopherols consumption. which are the most important natural antioxidants. The MO contained higher amount of total Acknowledgment tocopherols content (38.32 mg/100g) than that of M. A. Alim thanks the Swedish Institute (SI), RSO (25.57 mg/100g). The RSO contained 25.57 Box 7434, SE-103 91, Stockholm, Sweden, for mg/100g of total tocopherols which concur with supporting with fellowships. Z. Iqbal thanks the previously published results by Gunstone et al. Bangladesh Agricultural University, Mymensingh, (1994). All samples contained higher amounts of γ- Bangladesh, for his study leave. tocopherol than those of α- and δ-tocopherol. References Rancimat test Aaes-Jorgensen, E. 1972. Nutritional value of The oxidative stability is an important rapeseed oil, in Rapeseed Cultivation, parameter in ascertaining the quality of oils and Composition, Processing and Utilization, ed

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